Crystal Structure of PH0140: Exogenous Amino Acids Induce Open Octameric Assembly Enables Promoter^TTTT Binding for...

PH0140, a hypothetical protein from the Feast/Famine Regulatory Protein (FFRP) family in Pyrococcus horikoshii OT3, is predicted to play a role in transcriptional regulation in response to exogenous amino acids. Previous in-silico studies suggest that PH0140 has a binding preference for exogenous tryptophan and regulates transcription through DNA recognition and allostery. However, its structure and regulatory mechanism remain largely unexplored. In this study, we determined the crystal structure of PH0140 at a resolution of 2.0 Å, revealing that it binds to exogenous isoleucine through distinct structural features: a unique C-terminal loop region near the Effector-Binding Domain (EBD) and a β-strand (β2) with notable structural deviations from homologous proteins. Size-exclusion chromatography showed that PH0140 forms oligomers in the presence of exogenous isoleucine and tryptophan. Furthermore, the amino acids binding were characterized through isothermal titration calorimetry (ITC). Studies suggest that exogenous tryptophan has a better binding affinity than isoleucine. To explore the structural basis of this ligand effect, we modeled an octameric PH0140-DNA complex containing a 111-bp promoter fragment and performed multiple molecular dynamics simulations. The simulations revealed that the octameric assembly undergoes a conformational opening to interact with the DNA promoter^TTTT region. The hydrophobic driven force on the EBD results in distortion of β-strand (β4) into loop (Chain G) to adopt an open conformation, which facilitates interaction with the promoter^TTTT region for transcription regulation.